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The role of microRNA-145 in human embryonic stem cell differentiation into vascular cells
Atherosclerosis 219 (2011) 468–474
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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
The role of microRNA-145 in human embryonic stem cell differentiation into vascular cells Shintaro Yamaguchi a , Kenichi Yamahara a,b,∗ , Koichiro Homma a , Sayuri Suzuki a , Shizuka Fujii a , Ryuji Morizane a , Toshiaki Monkawa a , Yumi Matsuzaki c , Kenji Kangawa d , Hiroshi Itoh a a
Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan Department of Regenerative Medicine and Tissue Engineering, National Cerebral and Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan c Department of Physiology, Keio University School of Medicine, Tokyo, Japan d Department of Biochemistry, National Cerebral and Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan b
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Article history: Received 8 April 2011 Received in revised form 8 August 2011 Accepted 1 September 2011 Available online 9 September 2011 Keywords: Embryonic stem cells Vascular differentiation Smooth muscle cells microRNA-145
a b s t r a c t Background: Recent studies have reported that microRNA-145 (miR-145) is a critical mediator in the regulation of proliferation, differentiation, and phenotype expression of smooth muscle cells (SMCs). Previously, we established a system for differentiating human ESCs into vascular cells including endothelial cells (ECs) and vascular smooth muscle cells (SMCs). In the present study, we investigated the role of miR-145 in the differentiation process from human ESCs into ECs and SMCs. Methods and results: Undifferentiated human ESCs were induced to differentiate into vascular lineage according to our established method. Quantitative RT-PCR analysis revealed that human ESC-derived precursor of SMCs (ES-pre-SMCs), similar to human aortic SMCs, expressed a significant amount of miR145 as well as smooth muscle-specific proteins, compared to undifferentiated human ESCs, adult ECs, or ESC-derived ECs (ES-ECs). However, morphological analysis revealed that human ES-pre-SMCs appeared round and flattened in shape, though human aortic SMCs exhibited the typical spindle-like morphology of SMCs. In addition, Krüppel-like factor 4 and 5 (KLF4 and 5), direct targets of miR-145 and suppressors of smooth muscle differentiation, were upregulated in ES-pre-SMCs compared to aortic SMCs, indicating ES-pre-SMCs were not fully differentiated SMCs. Overexpression of miR-145 in ES-pre-SMCs upregulated the expression of smooth muscle markers, repressed KLF4 and 5 expressions, and changed their morphology into a differentiated spindle-like shape. Furthermore, by introduction of miR-145, ES-preSMC proliferation was significantly inhibited and carbachol-stimulated contraction of ES-pre-SMCs was significantly increased. In contrast, downregulation of miR-145 in ES-pre-SMCs upregulated KLF4 and 5 expressions, suppressed the expression of smooth muscle markers, and left unchanged their proliferation and contractility. In ES-ECs, miR-145 overexpression did not induce the synthesis of smooth muscle-related proteins nor suppress the expression of endothelial nitric oxide synthase. Conclusion: We showed that miR-145 can regulate the fate and phenotype of human ES-pre-SMCs as they become fully differentiated SMCs. Overexpression of miR-145 on human ES-pre-SMCs is a promising method to obtain functional mature SMCs from human ESCs, which are required for reliable experimental research in the fields of atherosclerosis, hypertension and other vascular diseases. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Vascular smooth muscle cells (SMCs) are a major component of cardiovascular systems and have been the subject of great interest in the field of cardiovascular diseases including atherosclerosis, hypertension, and vasospasm [1,2]. For basic/clinical research
∗ Corresponding author at: Department of Regenerative Medicine and Tissue Engineering, National Cerebral and Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. Tel.: +81 6 6833 5012; fax: +81 6 6835 5496. E-mail address: [email protected] (K. Yamahara). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.09.004
and drug discovery of these vascular smooth muscle disorders, establishing reliable differentiated vascular SMCs is essential. However, primary SMCs established from adult tissues are not terminally differentiated and undergo dedifferentiation from the contractile to the synthetic phenotype during cell culturing [3,4]. Recent studies indicate that embryonic stem cells (ESCs) are a promising cell source for SMCs [5–9]. Previously we demonstrated that human ESC-derived tumor rejection antigen 1-60 (TRA160) negative and vascular endothelial growth factor receptor-2 (VEGFR2) positive cells could differentiate into both ESC-
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derived endothelial cells (ECs) and SMCs [5,10]. Because human ESC-derived SMCs expressed major smooth muscle cytoskeletal proteins, we thought that these cells should belong to the smooth muscle lineage [10]. However, the expression of smooth muscle cytoskeletal proteins alone does not provide definitive evidence for differentiated SMCs, because mature SMCs possess distinct morphological characteristics and contractile functions [11]. Indeed, since our human ESC-derived SMCs did not present the typical spindle-like morphology of SMCs, we have called these cells human ESC-derived precursor of SMCs (ES-pre-SMCs). Recent studies have shown that microRNA-145 (miR-145) is strongly expressed in SMCs and regulates their differentiation [12,13]. In SMCs, miR-145 directly targets several key regulators of SMC phenotype including Krüppel-like factor 4, one of the core reprogramming to cell pluripotency factors and 5 (KLF4 and 5) and upregulates the expression of smooth muscle-specific genes including serum response factor and myocardin [12,13]. Therefore, miR-145 is a critical regulator of SMC fate and phenotype. In this study, we investigated the expression of miR-145 in our differentiation system of human ESC-derived vascular cells and its effect on the fate of endothelial or smooth muscle differentiation. We specifically determined whether introduction of miR-145 to human ES-pre-SMCs would result in the production of phenotypically and functionally mature SMCs in an attempt to establish a mature SMC line suitable for cardiovascular research, drug discovery, and cell therapy. 2. Materials and methods Detailed information is provided in online Supplementary material. 2.1. Cell culture Human ES-pre-SMCs and ESC-derived-ECs (ES-ECs) were obtained from human ESC lines KhES-1 and KhES-3 using a cell sorter (FACSMoFLo, Beckman Coulter, Brea, CA), as previously described [5,14,15]. These ESC-derived vascular cells and commercially available human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs) were used within six passages. 2.2. Transfections MiR-145 overexpression was induced by the transfection of 100 nM precursor microRNA-145 (pre-miR-145) (Ambion, Austin, TX) and its knockdown by 100 nM miR-145 inhibitor (anti-miR145) (Ambion, Austin, TX). Knockdown of KLF4 or KLF5 was accomplished using 100 nM siRNA (si-KLF4 and si-KLF5) (Qiagen, Valencia, CA). The transfected cells were cultured in differentiation medium for 48–72 h prior to study. 2.3. Immunocytochemistry Cultured cells were stained with various antibodies [7–10], as described in Supplemental Table 2. 2.4. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
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SYBR Green assay in all experiments showed a statistically significant difference in miR-145 expression. 2.5. Western blot analysis Western blotting was carried out using a standard protocol as described elsewhere [16]. The antibodies used are listed in Supplemental Table 4. 2.6. Cell proliferation assay Cell proliferation was assessed by (3-(4,5-dimethylthiazol2-yl)-2,5)-diphenyltetrazolium bromide (MTT) assay (NACALAI TESQUE, Japan) and bromodeoxyuridine (BrdU) incorporation assay (BD Pharmingen) as described previously [12,17]. 2.7. Contractility assay An agonist-induced contractility assay using carbachol (10 M) was performed as previously described [11]. 2.8. Statistical analysis Results are presented as means ± S.E.M. Comparisons of two populations were made by a two-tailed Student’s t test and comparisons among groups were tested by one-way ANOVA followed by the Tukey post hoc test. A P < 0.05 was considered significant. 3. Results 3.1. Differentiation of human ESCs towards vascular smooth muscle cells We induced differentiation of undifferentiated human ESCs (KhES-1 and KhES-3) to ES-pre-SMCs using our established method [5,10]. After culturing human ESCs with OP9 feeder cells for 10 days, we sorted TRA1-60- VEGFR2+ VE-cadherin− cells (about 2.5% of all cells, Fig. 1B, Supplemental Fig. 4B), which belong to mesodermal lineage other than ECs and should contain smooth muscle progenitor cells [18]. After culturing and several passages of sorted TRA1-60- VEGFR2+ VE-cadherin− cells, all these expanded cells lost VEGFR2 and expressed PDGFR- (Fig. 1D–F). 3.2. Characterization of human ES-pre-SMCs Immunostaining revealed that, similar to HASMCs, all expanded TRA1-60- VEGFR2+ VE-cadherin− cells were positive for smooth muscle specific cytoskeletal proteins including ␣-SMA, calponin, caldesmon and smooth muscle myosin heavy chain (SM-MHC) 1/2, as we reported previously (Fig. 1G, Supplemental Fig. 4C) [10]. Although HASMCs exhibited the typical spindle-like morphology of SMCs, almost all (>95%) of expanded TRA1-60- VEGFR2+ VEcadherin− cells appeared round and flattened in shape (Fig. 1G, Supplemental Fig. 4C), indicating these cells belong to the smooth muscle lineage, but might be at a rather immature stage compared to HASMCs. Therefore, we have called these cells “human ESC-derived precursor of SMCs (ES-pre-SMCs)”. 3.3. MiR-145 is abundant in human ES-pre-SMCs
Quantitative RT-PCR for miR-145 and mRNAs was performed using a miScript or QuantiFast SYBR Green PCR kit (Qiagen) with specific primers listed in Supplemental Table 3. Although a more than 2-fold difference in miR-145 expression was observed between the SYBR Green and TaqMan assays (data not shown), the
Quantitative RT-PCR demonstrated that miR-145 was abundant in human ES-pre-SMCs (9.1 ± 1.7-fold vs. ESCs, P < 0.01) and HASMCs (15.1 ± 1.6-fold vs. ESCs, P < 0.01), compared to human ECs or undifferentiated human ESCs (Fig. 1I). Among SMCs, miR-145
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Fig. 1. Expression of vascular markers and miR-145 in human ESC-derived vascular cells. (A–C) Flow cytometric analysis of cell surface markers on day 10 after the induction of differentiation in KhES-1. TRA1-60-negative and VEGFR2-positive differentiated cells accounted for about 6% of all cultured cells (A). VEGFR2+ VE-cadherin− cells constituted about 10% of TRA1-60-negative differentiated cells. These cells were sorted and used as human ES-pre-SMCs (B). (D–F) Flow cytometric analysis of cell surface markers of expanded ES-pre-SMCs. VEGFR2-positive cells accounted for less than 2% of all cells (D, E). PDGFR- positive cells constituted more than 70% of all cells (F). (G) Microscopic analysis of human ES-pre-SMCs. Both ES-pre-SMCs and control HASMCs were stained with antibodies for ␣-SMA, calponin, caldesmon, SM-MHC 1/2, and counterstained with DAPI. Scale bar: 100 m. (H) Western blot analysis of smooth muscle markers in ES-pre-SMCs and HASMCs. Expression profiles were similar in these cells. n = 4. (I) Quantitative analysis of miR-145 expression in undifferentiated human ESCs, ES-pre-SMCs, HASMCs, ES-ECs and HUVECs. n = 6, * P < 0.01.
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Fig. 2. Modulation of ES-pre-SMCs differentiation by overexpression or knockdown of miR-145. (A–C) The effect of pre-miR-145 and anti-miR-145 transfection (100 nM) on expression of smooth muscle-related molecules in human ES-pre-SMCs. Introduction of pre-miR-145 significantly increased (>3000-fold vs. mock, n = 3), whereas anti-miR145 decreased (0.16 ± 0.05-fold vs. mock, n = 6, * P < 0.05) the expression of miR-145 in ES-pre-SMCs (A). Transfection of pre-miR-145 increased but anti-miR-145 decreased gene expression of smooth muscle markers. n = 6. (B), as well as their protein expression. n = 4. (C, D). (* P < 0.05, ** P < 0.01 vs. mock-infected cells) (E–G). Western blot analysis of KLF4 and 5 in ES-pre-SMCs and HASMCs (n = 4, * P < 0.05 vs. ES-pre-SMCs) (E), pre-miR-145-transfected ES-pre-SMCs (n = 4, * P < 0.05 vs. mock-infected cells) (F), and antimiR-145-transfected ES-pre-SMCs (n = 4, * P < 0.05 vs. mock-infected cells) (G). (H) Effect of miR-145 modulation in human ES-pre-SMCs on gene expression of myocardin (n = 6, * P < 0.05, ** P < 0.01 vs. mock-infected cells). (I) The effect of KLF4 or KLF5 knockdown on the expression of myocardin and ␣-SMA in ES-pre-SMCs (n = 4, * P < 0.05 vs. non-targeted siRNA).
expression was slightly suppressed in ES-pre-SMCs compared to HASMCs (0.60 ± 0.2-fold, P < 0.01). 3.4. The effect of miR-145 modulation on human ES-pre-SMC differentiation The introduction of precursor miR-145 (pre-miR-145, 100 nM) elicited a greater than 3000-fold increase in miR-145 expression on human ES-pre-SMCs (Fig. 2A), and upregulated the expression of smooth muscle specific genes and proteins (Fig. 2B, C). In contrast, transfection of anti-miR-145 into human ES-pre-SMCs significantly decreased the expression of miR-145 (0.16 ± 0.05-fold, P < 0.05)
(Fig. 2A), as well as smooth muscle-specific genes and proteins (Fig. 2B, D). A recent study demonstrated that the contribution of miR-145 to SMC differentiation is mediated through the suppressed expression of its target gene KLF4 or KLF5 [12,13,19], which are potent negative regulators of transcriptional coactivator myocardin [12,13]. Both KLF4 and KLF5 were strongly expressed in human ES-preSMCs compared to HASMCs (KLF4; 1.45 ± 0.13-fold, P < 0.05, KLF5; 1.58 ± 0.19-fold, P < 0.05) (Fig. 2E), which might be associated with the immature state of ES-pre-SMCs. Introduction of pre-miR-145 to human ES-pre-SMCs significantly suppressed the expression of KLF4 and KLF5 proteins followed by upregulation of myocardin
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Fig. 3. Production of mature differentiated human ES-SMCs by miR-145 overexpression. (A) Representative morphological changes of ES-pre-SMCs after pre-miR-145 or antimiR-145 transfection confirmed by microscopy. Scale bar: 100 m. (B) Quantitative analysis of ES-pre-SMC proliferation by MTT assay. PDGF-BB-induced cell proliferation was significantly attenuated by miR-145 overexpression, but was not changed by miR-145 knockdown. n = 4, * P < 0.05. (C) The cell cycle analysis of ES-pre-SMCs by flow cytometry. MiR-145 overexpression decreased the number of G2/M phase. (D) Vasoconstrictor-induced contractile assay of ES-pre-SMCs. Overexpression of miR-145 augmented contractile response to carbachol (10 M), whereas knockdown of miR-145 resulted in unchanged contractility. (n = 6–7, * P < 0.05 vs. mock-infected cells). Scale bar 100 m. (E, F) Western blotting analysis of total and phosphorylated myosin light chain 2 (MLC2, Thr18/Ser19 and Ser19) in ES-pre-SMCs. Induction of pre-miR-145 (E) strengthened whereas knockdown of miR-145 (F) did not change the carbachol-induced increase in MLC2 phosphorylation (F) (n = 4, * P < 0.05 vs. mock-infected cells).
(Fig. 2F, H), whereas knockdown of miR-145 produced the opposite result. (Fig. 2G, H). 3.5. MiR-145 overexpression induced the differentiated contractile phenotype in human ES-pre-SMCs The introduction of miR-145 to human ES-pre-SMCs elicited a morphological change to a spindle-like shape that reflected a differentiated and contractile state (Fig. 3A). MTT assay revealed that transfection of pre-miR-145 significantly inhibited PDGF-BB-induced proliferation of human ES-pre-SMCs (miR145 overexpression: 1.22 ± 0.11-fold vs. control: 1.95 ± 0.06-fold, P < 0.05) (Fig. 3B), which was accompanied by a reduction of DNA synthesis (miR-145 overexpression: 4% of all cells vs. control: 10%) (Supplemental Fig. 3) and a reduction of the percentage of cells in the G2/M phase (miR-145 overexpression: 18.5% of all cells vs. control 22.4%) (Fig. 3C), confirmed by BrdU incorporation assay. A notable characteristic of differentiated SMCs is the ability to contract in response to vasoactive agents like carbachol [11,20]. Treatment with carbachol (10 M) produced a contraction
of 13.7 ± 2.4% in human ES-pre-SMCs (Fig. 3D, Supplemental movie 1) and pre-miR-145 treatment produced about double the peak contraction (Supplemental movie 2, 25.6 ± 3.7%, P < 0.05). In addition, pre-miR-145 overexpression significantly enhanced myosin light chain 2 (MLC2) phosphorylation in carbachol-treated human ES-pre-SMCs (phospho-MLC2 (Ser19) 1.57 ± 0.20-fold and phospho-MLC2 (Thr18/Ser19) 1.97 ± 0.34-fold, P < 0.05) (Fig. 3E). 3.6. MiR-145 overexpression did not alter the differentiation fate of human ES-ECs. Compared to human ES-pre-SMCs and HASMCs, miR-145 expression was markedly suppressed in both human ES-ECs and HUVECs (Fig. 1I). However, between these ECs, human ES-ECs showed increased miR-145 expression (2.3 ± 0.5-fold vs. HUVEC, P < 0.05) (Fig. 1I) and decreased expression of definite endothelial marker, endothelial nitric-oxide synthase (eNOS) (0.40 ± 0.03-fold vs. HUVEC, P < 0.05) (Fig. 4A), suggesting that human ES-ECs possess the ability to differentiate into smooth muscle cells. However, transfection of pre-miR-145 in human ES-ECs did not affect eNOS
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Fig. 4. Effect of miR-145 overexpression on human ES-ECs. (A) Representative western blotting of endothelial nitric-oxide synthase (eNOS) in hESCs, ES-ECs and HUVECs. n = 4. Both human ES-ECs and HUVECs expressed eNOS, though its expression was lower in human ES-ECs compared to HUVECs. (B, C) Western blotting analysis of eNOS and smooth muscle markers in pre-miR-145 overexpressed ES-ECs. n = 4. Transfection of pre-miR-145 in ES-ECs did not suppress eNOS expression (B) nor induced the expression of smooth muscle markers (C). * P < 0.05.
expression (Fig. 4B) nor induce the expression of smooth muscle markers (Fig. 4C).
4. Discussion In the present study, we demonstrated that human ESpre-SMCs expressed significant amounts of miR-145, a key regulator of smooth muscle differentiation, but its expression was slightly decreased compared to HASMCs. Similarly, compared with HASMCs, human ES-pre-SMCs were at a rather immature state of differentiation as determined by their morphological characteristics, smooth muscle-specific marker profiles, and contractile properties [10]. Introduction of miR-145 into human ES-pre-SMCs changed their morphology to a spindle-like shape, upregulated the expression of smooth muscle-specific markers, decreased proliferation, and increased the strength of carbachol-induced contraction. These results indicate that miR-145 can regulate the fate and phenotype of human ES-pre-SMCs into fully differentiated and contractile SMCs. Recent studies have indicated that miRNAs play a fundamental role in a wide range of cellular functions via degradation or translational inhibition of their target mRNAs [21,22]. MiR-145 is a 22 nucleotide miRNA highly conserved across vertebrates [23], and recent reports have suggested that the miR-145/miR-143 gene cluster is a novel phenotypic marker and modulator of vascular SMCs [12,24]. MiR-145/miR-143 knockout mouse had a thinner aortic wall thickness than the wild type mouse with dedifferentiated vascular SMCs [24]. Overexpression of miR-145 inhibited vascular neointimal formation and induced the
differentiation of vascular SMCs with the upregulation of smooth muscle differentiation markers [13]. In this study, we demonstrated that, similar to vascular SMCs, miR-145 was markedly expressed in human ES-pre-SMCs and introduction of miR-145 to ES-pre-SMCs changed their phenotype to a differentiated state. Recently, Xu et al. reported that miR-145 regulates the major pluripotency factors OCT4, SOX2, and KLF4 and represses pluripotency in human ESCs [22]. In undifferentiated ESCs, OCT4 represses transcription of miR-145, which leads to the upregulation of OCT4, SOX2, and KLF4. Therefore, miR-145 overexpression in undifferentiated ESCs might facilitate cell differentiation other than smooth muscle lineage. However, in this paper, we focus on the role of miR-145 in the differentiation process of ESC-derived SMCs towards mature SMCs. For this purpose, we prepared human ESC-derived smooth muscle precursor cells (ES-pre-SMCs), which already belong to the smooth muscle lineage, and investigated miR145-mediated effects on ES-pre-SMCs regarding SMC phenotype. In addition, Elia et al. reported that miR-145/-143 knockout mice only showed reduction in the smooth muscle layers of the aorta, indicating that manipulation of miR-145 in undifferentiated ESCs would not influence the phenotype of differentiated cells other than SMCs [24]. MiR-145 is reported to highly integrate into a transcriptional network and acts as a critical switch in smooth muscle differentiation [12]. Previous reports have noted that miR-145 targeted serum response factor, a key transcriptional factor for smooth muscle differentiation, and myocardin, its major transcriptional cofactor [12,13]. Furthermore, miR-145 suppresses targeted expression of several transcriptional factors including KLF4 and KLF5 and
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promotes differentiation of smooth muscle cells [12,13]. We demonstrated that KLF4 and KLF5 expressions were significantly upregulated in ES-pre-SMCs compared to HASMCs, suggesting an immature differentiation state of human ES-pre-SMCs. Introduction of miR-145 to ES-pre-SMCs markedly downregulated KLF4 and KLF5 expression with upregulation of myocardin and smooth muscle-specific markers. In addition, we confirmed both KLF-4 and KLF-5 independently regulate the expression of myocardin in our ES-pre-SMCs by their loss-of-function analysis. We previously demonstrated that ESC-derived VEGFR2-positive cells could differentiate into both ES-ECs and ES-pre-SMCs [5,10,14,15]. Therefore, we examined the possibility that miR-145 might induce the differentiation of human ES-ECs towards smooth muscle lineage. MiR-145 expression was ultimately suppressed in HUVECs, which was similar to previously reported results using primary vascular cells [13]. We found that miR-145 expression was obviously suppressed in human ES-ECs compared to SMCs, but slightly increased compared to HUVECs. Interestingly, consistent with the reduced expression of miR-145, KLF4 expression was upregulated in ES-ECs compared to ES-pre-SMCs (Supplemental Fig. 5). In addition, the expression of eNOS, a definite endothelial marker, was decreased in human ESECs compared to HUVECs, suggesting the premature differentiation state of the EC phenotype. However, transfection of pre-miR-145 in human ES-ECs did not induce the synthesis of smooth muscle markers nor affect eNOS expression. Therefore, our human ES-ECs would definitely be endothelial lineage-committed cells and not differentiate into vascular SMCs by miR-145 overexpression. In conclusion, we were able to establish morphologically and functionally mature human ESC-derived SMCs by the introduction of miR-145. Because primary SMCs are easy to dedifferentiate during cell culturing, fully differentiated human ES-SMCs are ideal SMCs for in vitro studies of biological, physiological, cellular, and molecular processes. Although previous reports including our papers described in detail the methods to induce SMCs from human ES cells [6,25], we, for the first time, have established a reliable strategy for the induction of a differentiated SMC line from ES cells. This cell line allows generation of an unlimited number of contractile SMCs, which are required for reliable experimental research in the fields of atherosclerosis, hypertension and other vascular diseases. Funding This work was supported by the Global Center of Excellence (GCOE) Program from the Ministry of Education, Culture, Sports, Science and Technology; Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology; Intramural Research Fund for Cardiovascular Diseases of National Cerebral and Cardiovascular Center; and a Banyu Foundation Research Grant sponsored by Banyu Life Science Foundation International. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis.2011.09.004.
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The role of microRNA-145 in human embryonic stem cell differentiation into vascular cells Shintaro Yamaguchi a , Kenichi Yamahara a,b,∗ , Koichiro Homma a , Sayuri Suzuki a , Shizuka Fujii a , Ryuji Morizane a , Toshiaki Monkawa a , Yumi Matsuzaki c , Kenji Kangawa d , Hiroshi Itoh a a
Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan Department of Regenerative Medicine and Tissue Engineering, National Cerebral and Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan c Department of Physiology, Keio University School of Medicine, Tokyo, Japan d Department of Biochemistry, National Cerebral and Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan b
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Article history: Received 8 April 2011 Received in revised form 8 August 2011 Accepted 1 September 2011 Available online 9 September 2011 Keywords: Embryonic stem cells Vascular differentiation Smooth muscle cells microRNA-145
a b s t r a c t Background: Recent studies have reported that microRNA-145 (miR-145) is a critical mediator in the regulation of proliferation, differentiation, and phenotype expression of smooth muscle cells (SMCs). Previously, we established a system for differentiating human ESCs into vascular cells including endothelial cells (ECs) and vascular smooth muscle cells (SMCs). In the present study, we investigated the role of miR-145 in the differentiation process from human ESCs into ECs and SMCs. Methods and results: Undifferentiated human ESCs were induced to differentiate into vascular lineage according to our established method. Quantitative RT-PCR analysis revealed that human ESC-derived precursor of SMCs (ES-pre-SMCs), similar to human aortic SMCs, expressed a significant amount of miR145 as well as smooth muscle-specific proteins, compared to undifferentiated human ESCs, adult ECs, or ESC-derived ECs (ES-ECs). However, morphological analysis revealed that human ES-pre-SMCs appeared round and flattened in shape, though human aortic SMCs exhibited the typical spindle-like morphology of SMCs. In addition, Krüppel-like factor 4 and 5 (KLF4 and 5), direct targets of miR-145 and suppressors of smooth muscle differentiation, were upregulated in ES-pre-SMCs compared to aortic SMCs, indicating ES-pre-SMCs were not fully differentiated SMCs. Overexpression of miR-145 in ES-pre-SMCs upregulated the expression of smooth muscle markers, repressed KLF4 and 5 expressions, and changed their morphology into a differentiated spindle-like shape. Furthermore, by introduction of miR-145, ES-preSMC proliferation was significantly inhibited and carbachol-stimulated contraction of ES-pre-SMCs was significantly increased. In contrast, downregulation of miR-145 in ES-pre-SMCs upregulated KLF4 and 5 expressions, suppressed the expression of smooth muscle markers, and left unchanged their proliferation and contractility. In ES-ECs, miR-145 overexpression did not induce the synthesis of smooth muscle-related proteins nor suppress the expression of endothelial nitric oxide synthase. Conclusion: We showed that miR-145 can regulate the fate and phenotype of human ES-pre-SMCs as they become fully differentiated SMCs. Overexpression of miR-145 on human ES-pre-SMCs is a promising method to obtain functional mature SMCs from human ESCs, which are required for reliable experimental research in the fields of atherosclerosis, hypertension and other vascular diseases. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Vascular smooth muscle cells (SMCs) are a major component of cardiovascular systems and have been the subject of great interest in the field of cardiovascular diseases including atherosclerosis, hypertension, and vasospasm [1,2]. For basic/clinical research
∗ Corresponding author at: Department of Regenerative Medicine and Tissue Engineering, National Cerebral and Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. Tel.: +81 6 6833 5012; fax: +81 6 6835 5496. E-mail address: [email protected] (K. Yamahara). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.09.004
and drug discovery of these vascular smooth muscle disorders, establishing reliable differentiated vascular SMCs is essential. However, primary SMCs established from adult tissues are not terminally differentiated and undergo dedifferentiation from the contractile to the synthetic phenotype during cell culturing [3,4]. Recent studies indicate that embryonic stem cells (ESCs) are a promising cell source for SMCs [5–9]. Previously we demonstrated that human ESC-derived tumor rejection antigen 1-60 (TRA160) negative and vascular endothelial growth factor receptor-2 (VEGFR2) positive cells could differentiate into both ESC-
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derived endothelial cells (ECs) and SMCs [5,10]. Because human ESC-derived SMCs expressed major smooth muscle cytoskeletal proteins, we thought that these cells should belong to the smooth muscle lineage [10]. However, the expression of smooth muscle cytoskeletal proteins alone does not provide definitive evidence for differentiated SMCs, because mature SMCs possess distinct morphological characteristics and contractile functions [11]. Indeed, since our human ESC-derived SMCs did not present the typical spindle-like morphology of SMCs, we have called these cells human ESC-derived precursor of SMCs (ES-pre-SMCs). Recent studies have shown that microRNA-145 (miR-145) is strongly expressed in SMCs and regulates their differentiation [12,13]. In SMCs, miR-145 directly targets several key regulators of SMC phenotype including Krüppel-like factor 4, one of the core reprogramming to cell pluripotency factors and 5 (KLF4 and 5) and upregulates the expression of smooth muscle-specific genes including serum response factor and myocardin [12,13]. Therefore, miR-145 is a critical regulator of SMC fate and phenotype. In this study, we investigated the expression of miR-145 in our differentiation system of human ESC-derived vascular cells and its effect on the fate of endothelial or smooth muscle differentiation. We specifically determined whether introduction of miR-145 to human ES-pre-SMCs would result in the production of phenotypically and functionally mature SMCs in an attempt to establish a mature SMC line suitable for cardiovascular research, drug discovery, and cell therapy. 2. Materials and methods Detailed information is provided in online Supplementary material. 2.1. Cell culture Human ES-pre-SMCs and ESC-derived-ECs (ES-ECs) were obtained from human ESC lines KhES-1 and KhES-3 using a cell sorter (FACSMoFLo, Beckman Coulter, Brea, CA), as previously described [5,14,15]. These ESC-derived vascular cells and commercially available human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs) were used within six passages. 2.2. Transfections MiR-145 overexpression was induced by the transfection of 100 nM precursor microRNA-145 (pre-miR-145) (Ambion, Austin, TX) and its knockdown by 100 nM miR-145 inhibitor (anti-miR145) (Ambion, Austin, TX). Knockdown of KLF4 or KLF5 was accomplished using 100 nM siRNA (si-KLF4 and si-KLF5) (Qiagen, Valencia, CA). The transfected cells were cultured in differentiation medium for 48–72 h prior to study. 2.3. Immunocytochemistry Cultured cells were stained with various antibodies [7–10], as described in Supplemental Table 2. 2.4. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
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SYBR Green assay in all experiments showed a statistically significant difference in miR-145 expression. 2.5. Western blot analysis Western blotting was carried out using a standard protocol as described elsewhere [16]. The antibodies used are listed in Supplemental Table 4. 2.6. Cell proliferation assay Cell proliferation was assessed by (3-(4,5-dimethylthiazol2-yl)-2,5)-diphenyltetrazolium bromide (MTT) assay (NACALAI TESQUE, Japan) and bromodeoxyuridine (BrdU) incorporation assay (BD Pharmingen) as described previously [12,17]. 2.7. Contractility assay An agonist-induced contractility assay using carbachol (10 M) was performed as previously described [11]. 2.8. Statistical analysis Results are presented as means ± S.E.M. Comparisons of two populations were made by a two-tailed Student’s t test and comparisons among groups were tested by one-way ANOVA followed by the Tukey post hoc test. A P < 0.05 was considered significant. 3. Results 3.1. Differentiation of human ESCs towards vascular smooth muscle cells We induced differentiation of undifferentiated human ESCs (KhES-1 and KhES-3) to ES-pre-SMCs using our established method [5,10]. After culturing human ESCs with OP9 feeder cells for 10 days, we sorted TRA1-60- VEGFR2+ VE-cadherin− cells (about 2.5% of all cells, Fig. 1B, Supplemental Fig. 4B), which belong to mesodermal lineage other than ECs and should contain smooth muscle progenitor cells [18]. After culturing and several passages of sorted TRA1-60- VEGFR2+ VE-cadherin− cells, all these expanded cells lost VEGFR2 and expressed PDGFR- (Fig. 1D–F). 3.2. Characterization of human ES-pre-SMCs Immunostaining revealed that, similar to HASMCs, all expanded TRA1-60- VEGFR2+ VE-cadherin− cells were positive for smooth muscle specific cytoskeletal proteins including ␣-SMA, calponin, caldesmon and smooth muscle myosin heavy chain (SM-MHC) 1/2, as we reported previously (Fig. 1G, Supplemental Fig. 4C) [10]. Although HASMCs exhibited the typical spindle-like morphology of SMCs, almost all (>95%) of expanded TRA1-60- VEGFR2+ VEcadherin− cells appeared round and flattened in shape (Fig. 1G, Supplemental Fig. 4C), indicating these cells belong to the smooth muscle lineage, but might be at a rather immature stage compared to HASMCs. Therefore, we have called these cells “human ESC-derived precursor of SMCs (ES-pre-SMCs)”. 3.3. MiR-145 is abundant in human ES-pre-SMCs
Quantitative RT-PCR for miR-145 and mRNAs was performed using a miScript or QuantiFast SYBR Green PCR kit (Qiagen) with specific primers listed in Supplemental Table 3. Although a more than 2-fold difference in miR-145 expression was observed between the SYBR Green and TaqMan assays (data not shown), the
Quantitative RT-PCR demonstrated that miR-145 was abundant in human ES-pre-SMCs (9.1 ± 1.7-fold vs. ESCs, P < 0.01) and HASMCs (15.1 ± 1.6-fold vs. ESCs, P < 0.01), compared to human ECs or undifferentiated human ESCs (Fig. 1I). Among SMCs, miR-145
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Fig. 1. Expression of vascular markers and miR-145 in human ESC-derived vascular cells. (A–C) Flow cytometric analysis of cell surface markers on day 10 after the induction of differentiation in KhES-1. TRA1-60-negative and VEGFR2-positive differentiated cells accounted for about 6% of all cultured cells (A). VEGFR2+ VE-cadherin− cells constituted about 10% of TRA1-60-negative differentiated cells. These cells were sorted and used as human ES-pre-SMCs (B). (D–F) Flow cytometric analysis of cell surface markers of expanded ES-pre-SMCs. VEGFR2-positive cells accounted for less than 2% of all cells (D, E). PDGFR- positive cells constituted more than 70% of all cells (F). (G) Microscopic analysis of human ES-pre-SMCs. Both ES-pre-SMCs and control HASMCs were stained with antibodies for ␣-SMA, calponin, caldesmon, SM-MHC 1/2, and counterstained with DAPI. Scale bar: 100 m. (H) Western blot analysis of smooth muscle markers in ES-pre-SMCs and HASMCs. Expression profiles were similar in these cells. n = 4. (I) Quantitative analysis of miR-145 expression in undifferentiated human ESCs, ES-pre-SMCs, HASMCs, ES-ECs and HUVECs. n = 6, * P < 0.01.
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Fig. 2. Modulation of ES-pre-SMCs differentiation by overexpression or knockdown of miR-145. (A–C) The effect of pre-miR-145 and anti-miR-145 transfection (100 nM) on expression of smooth muscle-related molecules in human ES-pre-SMCs. Introduction of pre-miR-145 significantly increased (>3000-fold vs. mock, n = 3), whereas anti-miR145 decreased (0.16 ± 0.05-fold vs. mock, n = 6, * P < 0.05) the expression of miR-145 in ES-pre-SMCs (A). Transfection of pre-miR-145 increased but anti-miR-145 decreased gene expression of smooth muscle markers. n = 6. (B), as well as their protein expression. n = 4. (C, D). (* P < 0.05, ** P < 0.01 vs. mock-infected cells) (E–G). Western blot analysis of KLF4 and 5 in ES-pre-SMCs and HASMCs (n = 4, * P < 0.05 vs. ES-pre-SMCs) (E), pre-miR-145-transfected ES-pre-SMCs (n = 4, * P < 0.05 vs. mock-infected cells) (F), and antimiR-145-transfected ES-pre-SMCs (n = 4, * P < 0.05 vs. mock-infected cells) (G). (H) Effect of miR-145 modulation in human ES-pre-SMCs on gene expression of myocardin (n = 6, * P < 0.05, ** P < 0.01 vs. mock-infected cells). (I) The effect of KLF4 or KLF5 knockdown on the expression of myocardin and ␣-SMA in ES-pre-SMCs (n = 4, * P < 0.05 vs. non-targeted siRNA).
expression was slightly suppressed in ES-pre-SMCs compared to HASMCs (0.60 ± 0.2-fold, P < 0.01). 3.4. The effect of miR-145 modulation on human ES-pre-SMC differentiation The introduction of precursor miR-145 (pre-miR-145, 100 nM) elicited a greater than 3000-fold increase in miR-145 expression on human ES-pre-SMCs (Fig. 2A), and upregulated the expression of smooth muscle specific genes and proteins (Fig. 2B, C). In contrast, transfection of anti-miR-145 into human ES-pre-SMCs significantly decreased the expression of miR-145 (0.16 ± 0.05-fold, P < 0.05)
(Fig. 2A), as well as smooth muscle-specific genes and proteins (Fig. 2B, D). A recent study demonstrated that the contribution of miR-145 to SMC differentiation is mediated through the suppressed expression of its target gene KLF4 or KLF5 [12,13,19], which are potent negative regulators of transcriptional coactivator myocardin [12,13]. Both KLF4 and KLF5 were strongly expressed in human ES-preSMCs compared to HASMCs (KLF4; 1.45 ± 0.13-fold, P < 0.05, KLF5; 1.58 ± 0.19-fold, P < 0.05) (Fig. 2E), which might be associated with the immature state of ES-pre-SMCs. Introduction of pre-miR-145 to human ES-pre-SMCs significantly suppressed the expression of KLF4 and KLF5 proteins followed by upregulation of myocardin
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Fig. 3. Production of mature differentiated human ES-SMCs by miR-145 overexpression. (A) Representative morphological changes of ES-pre-SMCs after pre-miR-145 or antimiR-145 transfection confirmed by microscopy. Scale bar: 100 m. (B) Quantitative analysis of ES-pre-SMC proliferation by MTT assay. PDGF-BB-induced cell proliferation was significantly attenuated by miR-145 overexpression, but was not changed by miR-145 knockdown. n = 4, * P < 0.05. (C) The cell cycle analysis of ES-pre-SMCs by flow cytometry. MiR-145 overexpression decreased the number of G2/M phase. (D) Vasoconstrictor-induced contractile assay of ES-pre-SMCs. Overexpression of miR-145 augmented contractile response to carbachol (10 M), whereas knockdown of miR-145 resulted in unchanged contractility. (n = 6–7, * P < 0.05 vs. mock-infected cells). Scale bar 100 m. (E, F) Western blotting analysis of total and phosphorylated myosin light chain 2 (MLC2, Thr18/Ser19 and Ser19) in ES-pre-SMCs. Induction of pre-miR-145 (E) strengthened whereas knockdown of miR-145 (F) did not change the carbachol-induced increase in MLC2 phosphorylation (F) (n = 4, * P < 0.05 vs. mock-infected cells).
(Fig. 2F, H), whereas knockdown of miR-145 produced the opposite result. (Fig. 2G, H). 3.5. MiR-145 overexpression induced the differentiated contractile phenotype in human ES-pre-SMCs The introduction of miR-145 to human ES-pre-SMCs elicited a morphological change to a spindle-like shape that reflected a differentiated and contractile state (Fig. 3A). MTT assay revealed that transfection of pre-miR-145 significantly inhibited PDGF-BB-induced proliferation of human ES-pre-SMCs (miR145 overexpression: 1.22 ± 0.11-fold vs. control: 1.95 ± 0.06-fold, P < 0.05) (Fig. 3B), which was accompanied by a reduction of DNA synthesis (miR-145 overexpression: 4% of all cells vs. control: 10%) (Supplemental Fig. 3) and a reduction of the percentage of cells in the G2/M phase (miR-145 overexpression: 18.5% of all cells vs. control 22.4%) (Fig. 3C), confirmed by BrdU incorporation assay. A notable characteristic of differentiated SMCs is the ability to contract in response to vasoactive agents like carbachol [11,20]. Treatment with carbachol (10 M) produced a contraction
of 13.7 ± 2.4% in human ES-pre-SMCs (Fig. 3D, Supplemental movie 1) and pre-miR-145 treatment produced about double the peak contraction (Supplemental movie 2, 25.6 ± 3.7%, P < 0.05). In addition, pre-miR-145 overexpression significantly enhanced myosin light chain 2 (MLC2) phosphorylation in carbachol-treated human ES-pre-SMCs (phospho-MLC2 (Ser19) 1.57 ± 0.20-fold and phospho-MLC2 (Thr18/Ser19) 1.97 ± 0.34-fold, P < 0.05) (Fig. 3E). 3.6. MiR-145 overexpression did not alter the differentiation fate of human ES-ECs. Compared to human ES-pre-SMCs and HASMCs, miR-145 expression was markedly suppressed in both human ES-ECs and HUVECs (Fig. 1I). However, between these ECs, human ES-ECs showed increased miR-145 expression (2.3 ± 0.5-fold vs. HUVEC, P < 0.05) (Fig. 1I) and decreased expression of definite endothelial marker, endothelial nitric-oxide synthase (eNOS) (0.40 ± 0.03-fold vs. HUVEC, P < 0.05) (Fig. 4A), suggesting that human ES-ECs possess the ability to differentiate into smooth muscle cells. However, transfection of pre-miR-145 in human ES-ECs did not affect eNOS
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Fig. 4. Effect of miR-145 overexpression on human ES-ECs. (A) Representative western blotting of endothelial nitric-oxide synthase (eNOS) in hESCs, ES-ECs and HUVECs. n = 4. Both human ES-ECs and HUVECs expressed eNOS, though its expression was lower in human ES-ECs compared to HUVECs. (B, C) Western blotting analysis of eNOS and smooth muscle markers in pre-miR-145 overexpressed ES-ECs. n = 4. Transfection of pre-miR-145 in ES-ECs did not suppress eNOS expression (B) nor induced the expression of smooth muscle markers (C). * P < 0.05.
expression (Fig. 4B) nor induce the expression of smooth muscle markers (Fig. 4C).
4. Discussion In the present study, we demonstrated that human ESpre-SMCs expressed significant amounts of miR-145, a key regulator of smooth muscle differentiation, but its expression was slightly decreased compared to HASMCs. Similarly, compared with HASMCs, human ES-pre-SMCs were at a rather immature state of differentiation as determined by their morphological characteristics, smooth muscle-specific marker profiles, and contractile properties [10]. Introduction of miR-145 into human ES-pre-SMCs changed their morphology to a spindle-like shape, upregulated the expression of smooth muscle-specific markers, decreased proliferation, and increased the strength of carbachol-induced contraction. These results indicate that miR-145 can regulate the fate and phenotype of human ES-pre-SMCs into fully differentiated and contractile SMCs. Recent studies have indicated that miRNAs play a fundamental role in a wide range of cellular functions via degradation or translational inhibition of their target mRNAs [21,22]. MiR-145 is a 22 nucleotide miRNA highly conserved across vertebrates [23], and recent reports have suggested that the miR-145/miR-143 gene cluster is a novel phenotypic marker and modulator of vascular SMCs [12,24]. MiR-145/miR-143 knockout mouse had a thinner aortic wall thickness than the wild type mouse with dedifferentiated vascular SMCs [24]. Overexpression of miR-145 inhibited vascular neointimal formation and induced the
differentiation of vascular SMCs with the upregulation of smooth muscle differentiation markers [13]. In this study, we demonstrated that, similar to vascular SMCs, miR-145 was markedly expressed in human ES-pre-SMCs and introduction of miR-145 to ES-pre-SMCs changed their phenotype to a differentiated state. Recently, Xu et al. reported that miR-145 regulates the major pluripotency factors OCT4, SOX2, and KLF4 and represses pluripotency in human ESCs [22]. In undifferentiated ESCs, OCT4 represses transcription of miR-145, which leads to the upregulation of OCT4, SOX2, and KLF4. Therefore, miR-145 overexpression in undifferentiated ESCs might facilitate cell differentiation other than smooth muscle lineage. However, in this paper, we focus on the role of miR-145 in the differentiation process of ESC-derived SMCs towards mature SMCs. For this purpose, we prepared human ESC-derived smooth muscle precursor cells (ES-pre-SMCs), which already belong to the smooth muscle lineage, and investigated miR145-mediated effects on ES-pre-SMCs regarding SMC phenotype. In addition, Elia et al. reported that miR-145/-143 knockout mice only showed reduction in the smooth muscle layers of the aorta, indicating that manipulation of miR-145 in undifferentiated ESCs would not influence the phenotype of differentiated cells other than SMCs [24]. MiR-145 is reported to highly integrate into a transcriptional network and acts as a critical switch in smooth muscle differentiation [12]. Previous reports have noted that miR-145 targeted serum response factor, a key transcriptional factor for smooth muscle differentiation, and myocardin, its major transcriptional cofactor [12,13]. Furthermore, miR-145 suppresses targeted expression of several transcriptional factors including KLF4 and KLF5 and
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promotes differentiation of smooth muscle cells [12,13]. We demonstrated that KLF4 and KLF5 expressions were significantly upregulated in ES-pre-SMCs compared to HASMCs, suggesting an immature differentiation state of human ES-pre-SMCs. Introduction of miR-145 to ES-pre-SMCs markedly downregulated KLF4 and KLF5 expression with upregulation of myocardin and smooth muscle-specific markers. In addition, we confirmed both KLF-4 and KLF-5 independently regulate the expression of myocardin in our ES-pre-SMCs by their loss-of-function analysis. We previously demonstrated that ESC-derived VEGFR2-positive cells could differentiate into both ES-ECs and ES-pre-SMCs [5,10,14,15]. Therefore, we examined the possibility that miR-145 might induce the differentiation of human ES-ECs towards smooth muscle lineage. MiR-145 expression was ultimately suppressed in HUVECs, which was similar to previously reported results using primary vascular cells [13]. We found that miR-145 expression was obviously suppressed in human ES-ECs compared to SMCs, but slightly increased compared to HUVECs. Interestingly, consistent with the reduced expression of miR-145, KLF4 expression was upregulated in ES-ECs compared to ES-pre-SMCs (Supplemental Fig. 5). In addition, the expression of eNOS, a definite endothelial marker, was decreased in human ESECs compared to HUVECs, suggesting the premature differentiation state of the EC phenotype. However, transfection of pre-miR-145 in human ES-ECs did not induce the synthesis of smooth muscle markers nor affect eNOS expression. Therefore, our human ES-ECs would definitely be endothelial lineage-committed cells and not differentiate into vascular SMCs by miR-145 overexpression. In conclusion, we were able to establish morphologically and functionally mature human ESC-derived SMCs by the introduction of miR-145. Because primary SMCs are easy to dedifferentiate during cell culturing, fully differentiated human ES-SMCs are ideal SMCs for in vitro studies of biological, physiological, cellular, and molecular processes. Although previous reports including our papers described in detail the methods to induce SMCs from human ES cells [6,25], we, for the first time, have established a reliable strategy for the induction of a differentiated SMC line from ES cells. This cell line allows generation of an unlimited number of contractile SMCs, which are required for reliable experimental research in the fields of atherosclerosis, hypertension and other vascular diseases. Funding This work was supported by the Global Center of Excellence (GCOE) Program from the Ministry of Education, Culture, Sports, Science and Technology; Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology; Intramural Research Fund for Cardiovascular Diseases of National Cerebral and Cardiovascular Center; and a Banyu Foundation Research Grant sponsored by Banyu Life Science Foundation International. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis.2011.09.004.
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